Process for purifying neomycin



Patented Aug. 17, 1954 2,686,749 a o -arrest PR ocEsslFon BURIFYINGNEOMYCINI- lierbei t ll.":Wehrmcister, Terre Haute; Ind a, as-

s'ignor "to Commercial Solvents "Gorpom'tion,'1T:err,eiI-Iaute;,lI1id.=,-ra:corporaltion oiliMamylanl No Drawing.

streptomycin-but is--=active* against many'streptQ- myc-in-resistantstrains I (if-bacteria including M. tuberculosis.

Necmycin is produced by a soil" "organism of the fitreptomycesspeciessimilar to streptomyees jradiae when g'rown in nutr-i'ent media; Theneo' mycin 1 thus 1 produced 1 F is a thermostable, basic compoundwhich-is soluble inwater. Because of In the "past; neomycin has 'beemrecovered from the culture media in Wh-ichZit-has been' produceti by avariety of processes; licwever; the "mad orityof these processesproduce- 'ne'cmycinof :a low 1 purity; It thereforelias- 3 becomenecessary *to' find a methoil fon'tlieTurtl-ienpurification of-neomycinalready-recovered -inan impure form-fromthe cultur media inwhich it hasbeen produced;

I have :now discovered l a process Tor purifying neomycin which"consistsprimarily imformirrg an intermediate salt of neomycin withan-thraquinone+fl-sulfonicaid and subsequent-ly cleaving the neomycinanthraquinonefi sulfonatw with a strong: acid which forms -a neutralow-acid saw of neomycin of high: potency. By thus empl'oying my newprocess, I am able to take recovered neomycin of aZl'owmrdenof-purity,containing-at least about 50* unitsper milligram, the-'neomycin unitziasdefined by Waksman -bei'ng the minimum amount of neomycin "-that w llcompletely i inhibit: the growth of' Esclterichia ccli ATCC' 963'? in,one f m1; ofx nutrient agar, and raise the potency to azva'lu in"excess-of abouti2U0 units per "milligram thus largely eliminating toxiccomponents of the impure neor nycin and rend'ering it sui tiable fortherapeutic use.

:As *I' 'have previously mentioned abcve; the

first step: off: my tprocess consists of 'forming a and; 50

isolating the salt, neomycin 'anthraquinonewsulfonate. neom ycin iisw,hase; aiti will react with anthraquinone-ls sulfonic acid: to formasthesaltlveryrreani-ly: IHowever theanthraquinone -5 8 '-.=su'lfoni'c acid?"is most rwdllymavail abledn-theu'orm: of its: sodium'saltand ithereforeI" reactwthe neomycin wi th soui'umanthraquinoneefisulfonatezinzthezpresenc zofiiazmacidzistmng encughttodisplaceithe,anthraqumoneeeasulfouic acid fmmcthexsaltzform;:Stmngzmineral acids 'Applicationilleceniber 5,195.0; JSeriaII'NO.199341" BrfGlaimsa. (o1. s eve-c5) such as sulfuric, hydrochloric, andnitric acidi are particularly-- suitable for this phase 0f my processand can ti -employed with equal --efiec-- tiven ess with other-alka1i=metal* or alkaline earth metal salts of anthraquinone -;fl-sulfonicacid "if such salts "are more readily available rthan the usual sodiumsalt;

In carrying out this step of my process; I- employ at 1 least one=sequiya-lcnt 'weight oI "-anthrxat if quinone=B sulfonicaeidg or thealkali or'al'kal'ine earth metal salts, such as the sodium salt-as I thecase-may be; *pereq-uivalent weight *dfithe basic reactivegroups Qf'neomycin. Whenwan alkalior'al'kaline earth metal salt-=such as thesodiumployed, I addmne equivalent=weight= o'f the strong acid, asmentioned above "I have f-cuntlthat when enough acid *is' edtled -sothat the pH is lowered to'about GEO-anti below, "about one-equivalent ofthe *strong -acid hasbeen "added re-- suiting inthe ionizaticnmf theant'l'lraquinonec-surmnic acid-and 'the 'cation componentof th -salt"employed; 'Upon *carrying 'out this reaction=neomycin 'anthraquinone flsulfonatepre cipitatesf from the reacti-onmixture andthe pre cipitatemay be collected in any suitable man-- ner such as'forexampleby--filtrat-ion.

Neomycin anthraquinone-B-sulfonateis a yel low needle likwcrystallinemateri al which is crystallizablefrom "a suitable --s0lvent' SUGhZES a"water and alcohcl mixture; Neomycin' anthraquinone fi sul fonatc is"optically active andrgen eral'l y, has 'a neomyc'in activityoffront-about 75* toabout fi units per milligram:

Following the preparation" and isolation of' neomycin--anthraquinoneq8=sulfonatm the next step immynnew pi'ccessis to cleavethe anthraquinone B-sulfonic acid radical 1 from the neomycin compcnentofthe salt by dissolving the crystalline necmycin -arnthraquinone-psulfonate ir'rarr aquecue-solution of a strcng mineral witl such as for"examplesul'f'ui-ic' acicl, li yilroch-loricacidj-etc. The resultof' thisc'leavage is=to formr a *saltmfneomycin with the' anicmof the=particularacid-employetl; Here l! employ -one--' e quivalent' We-igl'it of theacidper' equivalent of the-neomycin antl'n'aquincne fi sulfonate and 'l"prefer to usean excess o'f "tlie"aci=d-in-ortlerto insure substantially complete replacement oftheantlirwquinone-fi-sulfonicacidcomponent of the neomycin anthraquinone-B sulfonate and, in turn,subsequent; substantially 'completer precipitation of: thessaltmft'neomycmslwith ether-anion; off-the: acid' used; Thelneswasaltewhichisutfonncclazmay be; arneutraliorcanuacitlusait:tlependingaupnnsthe:acidrguscd; Floreexample,neomycinacidcsuliiateiisn fnrmedgmhen sulfuric:acid lis .employeclxtorcleave. thec'neomycin:anthraqnmone.-.fi.+'sulfionate:

ilThe ineomycinisalticr acid sait, such'as mecmyer61)! cm'racidsulfate:ohtamedrsasiahovecdescribedisis? next precipitated from the acidsolution by the addition of a water soluble lower aliphatic: alcoholuntil substantially all of the neomycin salt or acid salt isprecipitated. Generally, I prefer to use about 8 to 10 volumes ofalcohol per volume of aqueous acid solution containing the neomycin saltor acid salt in order to insure substantially complete precipitation ofthe neomy cin salt or acid salt, which is then recovered by filtrationor any other suitable means. i. If the 7 ml; of methanol.

4 to yield 427 grams of neomycin anthraquinonefi sulfonate containing'76: units of neomycin per milligram (total of 32.5 million units) Theneomycin anthraquinone-B-sulfonate was dissolved in 4700 ml. of waterwith 352 ml. of concentrated sulfuric acid and this solution poured into47,000 Precipitated neomycin acid sulfate weighing 146 gms. was filteredand found precipitated neomycin salt is a neutral salt itmay berecovered and flied without further processing; However, if theprecipitated salt is an acid salt,

to contain 176 units of neomycin per milligram (total of 27 millionunits). The neomycin acid sulfate was then dissolved in water and thesolution passed through a column containing a such as, for example,neomycin acid sulfate. it I the excess acid sulfate such as an alkalineearth.

metal hydroxide. In carrying out the neutralization steps, the alkalineearth metal hydroxide is added until the pH of the mixture reaches '7.The neutralization can as well be carried out.

by passing the neomycin acid salt .through a strong basic anion exchangeresin converting the acid salt to the free neomycin base. The pH of thefree neomycin base is then adjusted to 7.0

calcium hydroxide. When a different acid salt is precipitated it is, ofcourse, necessary to use a base which forms an insoluble salt with theacid and which does not-react with or otherwise harmfully affect theneomycin or salt thereof under the conditions of use. When the neomycinacid salt has been neutralized by any of the methods shown above, theremaining liquid is an aqueous solution of a neomycin neutral salt fromwhich the neomycin neutral salt is readily recovered. The water can beremoved from this solution by any suitable means such as by spray dryingor by vaporization of the water under vacuum while in the frozen state,or the neomycin neutral salt can be precipitated by adding a watersoluble lower aliphatic alcohol to the aqueous solution.

The following examples are offered to illustrate my invention but theinvention is not to be considered as limited to the examples as shown.The scope of my invention is defined by this specification and theattached claims and obvious equivalents of the materials and operationsshown are to be considered as lying within my disclosure.

Example I A 1559 m1. portion of an aqueous solution of impure neomycincontaining 21,900 units of neomycin per m1. (total of 34 million units)was added with stirring to a solution of 457 grams of sodiumanthraquinone-/3-sulfonate in 14.5 liters of water and the pH of theresulting solution was. adjusted to 3.0 with hydrochloric acid. A pre--cipitate formed and was filtered, washed with water and recrystallizedfrom aqueous ethanol strong basic, anion-type, ion-exchange resin toobtain the neomycin free base. The resin is a high molecular,water-insoluble, quaternary ammonium hydroxide. The resin is sold underthe trade name of Amberlite XE-75 by the Rohm and Haas Company. The pHof the solution of neomycin free base was then adjusted to 7.0 withsulfuric acid and the neutral solution was then freeze dried undervacuum to yield 104 gms.

of neomycin sulfate containing 214 units of neo-,

mycin per milligram (total of 22.25 million units).

Example II I An 1800 ml. portion of an aqueous solution of sodiumanthraqumonee-sulfonate in 14.5 liters.

of water and the pH adjusted to 3.0 with hydrochloric acid. Aprecipitate formed and this was filtered, washed with water andrecrystallized from aqueous ethanol to yield 366 gms. of neomycinanthraquinone-p-sulfonate containing 81 units of neomycin per milligram(total of 30 million units). The neomycin anthraquinone-{isulfonate wasdissolved in 3900 ml. of water with 291 ml. of concentrated sulfuricacid and this solution poured into 39,000 ml. of methanol. Precipitatedneomycin acid sulfate weighing 138 gms. was filtered and found tocontain 181 units per milligram (total of 25,million units) portion ofthis neomycin acid sulfate was then dissolved in water and neutralizedbyadjusting the pH to 7.0 with calcium hydroxide. The precipitate whichformed was removed and the remaining solution freeze dried under vacuumto yield 65 grams of neutral neomycin sulfate containing 218 units ofneomycin per milligram.

Now having disclosed my invention what I claim is: 1

l. A process for purifying neomycin produced by culturing Streptomycesjradiae on liquid nu-' trient media which comprises mixing an impureaqueous solution of neomycin with an aqueous solution ofanthraquinone-p-sulfonic acid, collecting precipitated neomycinanthraquinone-Br sulfonate; dissolving the neomycinanthraquinone-fi-sulfonate in an aqueous solution of a mineral acidselected from the group consisting of sulfuric, hydrochloric, and nitricacid, adding the.

resulting solution to a water miscible lower aliphatic alcohol, andcollecting the precipitated neomycin salt which forms. 1

2. A process for purifying neomycin produced by culturing Streptomycesfradiae on liquid nutrient media which comprises mixing one equivalentof the basic reactive groups of neomycin with one equivalent of sodiumanthraquinone-psulfonate, adjusting the pH to below about 5.0 withhydrochloric acid, collecting precipitated neomycinanthraquinone-p-sulfonate, recrystallizing neomycinanthraquinone-cssulfonate, dis- A 93 gramsolving one equivalent ofneomycin anthraquinone-,c-sulfonate in an aqueous solution of oneequivalent of sulfuric acid, adding the resulting solution to methanol,collecting precipitated neomycin acid sulfate, dissolving neomycin acidsulfate in water, neutralizing the aqueous solution. of neomycin acidsulfate with calcium hydroxide, removing precipitated calcium sulfateand subsequently drying the remaining solution from the frozen state andrecovering dry solid neomycin sulfate.

3. A process for the purification of neomycin produced by culturingStreptomyces ,"fradiae on liquid nutrient media which comprises mixingan aqueous solution of one equivalent of the basic reactive groups ofneomycin with one equivalent of anthraquinone-fi-sulfonic acid,collecting precipitated neomycin anthraquinone-c-sulfonate, dissolvingone equivalent of neomycin anthraquinone-p-sulfonate in an aqueoussolution of one equivalent of sulfuric acid, adding the resultingsolution to methanol, collecting precipitated neomycin acid sulfate,dissolving neomycin acid sulfate in water, passing the aqueous solutionof neomycin acid sulfate through a strongly basic; anion exchange resin,adjusting the pH of said solution to 7.0 with sulfuric acid, addingmethanol to said solution and recovering precipitated neomycin sulfatewhich forms.'

4. A process for purifying neomycin produced by culturing Streptomycesfmdiwe on liquid nutrient media which comprises mixing an impure aqueoussolution of the said neomycin with an aqueous solution of an alkalimetal salt of anthraquinone-c-sulfonic acid, collecting precipitatedneomycin anthraquinone-fi-sulfonate, dissolving the neomycinanthraquinone-p-sulfonate in an aqueous solution of a mineral acidselected from a group consisting of sulfuric, hydrochloric, and nitricacid, adding the resulting solution to a Water miscible lower aliphaticalcohol, and collecting the precipitated neomycin salt which forms.

5. A process for purifying neomycin produced by culturing Streptomycesfradzae on liquid nutrient media which comprises mixing one equivalentweight of an alkali metal salt of anthraquinone-p-sulfonic acid with oneequivalent weight of the basic reactive groups of said aqueous neomycinin impure form, adjusting the pH to below about 5.0 with a strongmineral acid selected from the group consisting of sulfuric, hy-

drochloric, and nitric acid, collecting the precipitated neomycinanthraquinone-c-sulfonate, dissolving one equivalent Weight of theneomycin anthraquinone-,8-sulfonate in an aqueous solution of at leastone equivalent weight of a mineral acid selected from the groupconsisting of sulfuric, hydrochloric, and nitric acid, adding theresulting solution to a water miscible lower aliphatic alcohol, andcollecting the precipitated neomycin salt which forms.

6. The process of claim 5 wherein the alkali metal salt ofanthraquinone-B-sulfonic acid is sodium anthraquinone-p-sulfonate.

7. The process of claim 5 wherein the strong mineral acid used to adjustthe pH to below about 5.0 is hydrochloric acid.

8. A process for purifying neomycin produced by culturing Streptomycesfradiae on liquid nutrient media which comprises mixing one equivalentweight of the basic reactive groups of the said aqueous neomycin inimpure form with one equivalent Weight of an alkali metal salt ofanthraquinone-c-sulfonic acid, adjusting the pH to below about 5.0 withhydrochloric acid, collecting precipitated neomycinanthraquinone-psulfonate, dissolving one equivalent weight ofanthraquinone-s-sulfonate in an aqueous solution of at least oneequivalent weight of sulfuric acid, mixing the resulting solution withmethanol, collecting precipitated neomycin acid sulfate, neutralizingthe neomycin acid sulfate, and recovering the neomycin sulfatetherefrom.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 2,462,175 Folkers Feb. 22, 1949 2,501,014 Wintersteiner et a1.Mar. 21, 1950 2,516,080 Sobin et al. July 18, 1950 2,537,934 Lott Jan.9, 1951 OTHER REFERENCES Swart et al., article in Archives of Biochem,Nov. 1949, vol. 24, pages 92-103.

Peterson et al., article in J. A. C, 8., vol. '72, published 1950, pages3598-3603.

Peck, letter in J. A. C. 8., pages 2590 and 2591, July 2949.

Barron, Handbook of Antibiotics. pages 171- 174, published 1950,Reinhold Publishing 00., N. Y. C.

1. A PROCESS FOR PURIFYING NEOMYCIN PRODUCED BY CULTURING STREPTOMYCESFRADIAE ON LIQUID NUTRIENT MEDIA WHICH COMPRISES MIXING AN IMPUREAQUEOUS SOLUTION OF NEOMYCIN WITH AN AQUEOUS SOLUTION OFANTHRAQUINONE-B-SULFONIC ACID, COLLECTING PRECIPITATED NEOMYCINANTHRAQUINONE-BSULFONATE, DISSOLVING THE NEOMYCINANTHRAQUINONE-B-SULFONATE IN AN AQUEOUS SOLUTION OF A MINERAL ACIDSELECTED FROM THE GROUP CONSISTING OF SULFURIC, HYDROCHLORIC, AND NITRICACID, ADDING THE RESULTING SOLUTION TO A WATER MISCIBLE LOWER ALIPHATICALCOHOL, AND COLLECTING THE PRECIPTATED NEOMYCIN SALT WHICH FORMS.